To establish the epidemiological cut-off values (ECOFFs) for cefoselis against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Proteus mirabilis and Pseudomonas aeruginosa.We collected 2288 non-repetitive clinical isolates from five laboratories throughout four cities in China. The cefoselis MICs and inhibition zone diameters for all isolates were established using the broth microdilution method and the disc diffusion method following EUCAST guidelines. MIC ECOFFs were determined by visual estimation and ECOFFinder software. Zone diameter ECOFFs were set if a high correlation of MICs and inhibition zone diameters was found by Pearson correlation. Zone diameter ECOFFs were finally determined by the visual estimate method.MICs of cefoselis were distributed from 0.008 to >256 mg/L for the four Enterobacterales species and from 0.25 to >256 mg/L for P. aeruginosa. MIC ECOFFs were 0.125 mg/L for E. coli, K. pneumoniae and P. mirabilis, 0.25 mg/L for E. cloacae and 32 mg/L for P. aeruginosa. A high correlation of MICs and zone diameters was observed for all Enterobacterales (|r| > 0.8, P < 0.001) and a relatively high correlation was found for P. aeruginosa (|r| = 0.71, P < 0.001). The zone diameter ECOFF was 24 mm for E. cloacae, E. coli and K. pneumoniae, 26 mm for P. mirabilis and 21 mm for P. aeruginosa.We determined MIC and zone diameter ECOFFs for cefoselis against four Enterobacterales species and P. aeruginosa. The establishment of ECOFFs for cefoselis provides clinicians with helpful guidance to differentiate WT and non-WT pathogens.© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.